阻断细胞信号传导子和转录激活子6基因表达对人结肠癌细胞凋亡的影响  

Impact of STAT6 signaling pathway blockade on apoptosis of human colon cancer cells

作　　者：张明生[1] 周云峰[1] 谢丛华[1] 张文杰[1] 周福祥[1] 屈雪菊[2] 

机构地区：[1]武汉大学中南医院肿瘤放化疗科,430071 [2]武汉大学医学结构生物学研究中心

出　　处：《中华医学杂志》2006年第2期76-81,共6页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30470981);湖北省自然科学基金资助项目(2005ABA103)

摘　　要：目的探讨细胞信号传导子和转录激活子6(STAT)信号传导通路与结肠癌细胞凋亡的关系。方法构建4个STAT6特异的shRNA质粒载体,应用阳离子脂质体瞬时转染人结肠癌细胞HT-29,以阻断Stat6信号通路;分别用逆转录聚合酶链反应(RT-PCR)方法检测STAT6mRNA表达和运用流式细胞术检测磷酸化STAT6蛋白(pSTAT6)表达来筛选有效的小发夹RNA(shRNA);运用流式细胞术分析细胞凋亡变化;RT-PCR和Western印迹观察Bcl-2和Bax基因表达。结果成功构建并筛选出两个STAT6特异的shRNA质粒载体;STAT6基因沉默的结肠癌细胞中凋亡细胞明显增加,空白对照组细胞早期凋亡率为0.4%,pshRNA-STAT6-1组和pshRNA-STAT6-4组分别为13.0%和8.8%(P<0.05);Stat6信号通路被阻断的结肠癌细胞中Bcl-2基因表达明显减少,而Bax蛋白基因表达明显增多(P<0.01)。结论Stat6信号传导通路能抑制结肠癌细胞凋亡,可能是通过调节Bcl-2和Bax基因的表达发挥作用。Objective To investigate the relationship between the signal transducer and activator of transcription 6 （STAT6） and human colon cancer. Methods Four STAT6-specific recombinant plasmid vectors, pshRNA-STAT6-1, 2, 3, and 4 were constructed and transfected into the cultured human colon cancer cells of the line HT-29. Seventy-two hours later RT-PCR was used to detect the mRNA expression of STAT6 and the apoptosis-related genes Bcl-2 and Bax, flow cytometry （FCM） was used to detect the protein expression of phopho-STAT6 （pSTAT6）. HT-29 cells were inoculated into a plate and transfected with pshRNA-STAT6-1 or pshRNA-STAT6-4, and HT-29 cells without transfection were used as controls. Seventy-two hours later FCM was used to observe the cell apoptosis. Another HT-29 cells were inoculated into a plate and transfected with pshRNA-STAT6-1 or pshRNA-STAT6-4, or blank liposome as controls. Seventy-two hours later. Western blotting was used to detect the protein expression of Bcl-2 and Bax genes. Results The p-STAT6 protein expression rate was 3. 2%±0. 6% in the pshRNA-STAT6-1 group, significantly lower than that of the blank control group （18.2%±0.9% , P 〈 0.01 ） with an inhibition rate of 82.4%, and was 7.9%-0.4% in the pshRNA-STAT6-4 group, significantly lower than that in the blank control group too （P〈0.01 ） with an inhibition rate of 56.6%. And the p-STAT6 protein expression rates of the pshRNA-STAT6-2 and pshRNA-STAT6-3 groups were 16.6%±0.5% and 17. 1%±0.7% respectively, both not significant different from that of the blank control group （ both P 〉 0.05 ）. The early cell apoptosis rates of the pshRNA-STAT6-1 and pshRNA-STAT6-4 groups were 13. 0% and 8. 8% respectively, both significantly higher than that of the blank control group （ 0.4%, both P 〈 0.05 ）. The mRNA expression of Bcl-2 was significantly lower and the mRNA expression of Bax was significantly higher in the pshRNA-STAT6-1 and pshRNA-STAT6-4 groups than in the blank control and blank liposome groups （all P 〈0.01 �

关 键 词：信号传递 结肠肿瘤 SHRNA 脱噬作用 

分 类 号：R735.35[医药卫生—肿瘤]